Compositions and Methods for Inhibiting Entry of a Hepatic Virus

ABSTRACT

The present invention embraces Niemann-Pick C1-like 1 protein antagonists and agents that inhibit hepatic virus infection for use in the prevention and treatment of a hepatic virus infection.

INTRODUCTION

This application claims the benefit of priority of U.S. ProvisionalApplication Nos. 61/169,899, filed ApR. 16, 2009 and 61/093,549, filedSep. 2, 2008, the contents of which are incorporated herein by referencein their entireties.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV), a positive-strand RNA flavivirus, is a leadingcause of liver disease worldwide. Although acute infection can beasymptomatic, approximately 80% of patients fail to clear the virusresulting in a chronic infection associated with significant liverdisease, including steatosis, insulin-resistance, fibrosis, cirrhosisand hepatocellular carcinoma (Alter & Seeff (2000) Semin. Liver Dis.20:17-35). As such, HCV-related cancer accounts for over 50% ofhepatocellular carcinoma cases and over 30% of liver transplantsperformed in the United States. Despite this obvious public healthburden, there is no vaccine to prevent infection.

HCV is thought to enter cells via receptor-mediated endocytosisbeginning with binding of the viral particle to a series of cell surfacereceptors, including the tetraspanin CD81 (Pileri, et al. (1998) Science282:938-41), the scavenger receptor class B member I (SR-BI) (Scarselli,et al. (2002) EMBO J. 21:5017-25) and the tight-junction proteinsclaudin-1 (Evans, et al. (2007) Nature 446:801-5) and occludin (Liu, etal. (2009) J. Virol. 83:2011-4; Ploss, et al. (2009) Nature 457:882-6),followed by clathrin-mediated endocytosis (Blanchard, et al. (2006) J.Virol. 80:6964-72; Meertens, et al. (2006) J. Virol. 80:11571-8) andclass II fusion (Garry & Dash (2003) Virology 307:255-65) between thevirion envelope and the endosomal membrane. Additionally, thelow-density lipoprotein receptor (LDLR) (Agnello, et al. (1999) Proc.Natl. Acad. Sci. USA 96:12766-12771; Monazahian, et al. (1999) J. Med.Virol. 57:223-229; Wunschmann, et al. (2000) J. Virol. 74:10055-10062),asialoglycoprotein receptor (Saunier, et al. (2003) J. Virol.77:546-559), protocadherin β5 (Womg-Staal, et al. (2008) 15thInternational Symposium on Hepatitis C Virus & Related Viruses. SanAntonio, Tex.), and glycosaminoglycans (heparan sulfate) (Barth, et al.(2003) J. Biol. Chem. 278:41003-41012; Barth, et al. (2006) J. Virol.80:10579-10590; Bartosch, et al. (2003) J. Exp. Med. 197:633-642) havebeen implicated; however, the role of those agents has not beenconclusively proven to be essential for HCV entry. In addition, it hasbeen shown that the HCV particle is not only uniquely enriched incholesterol (Aizaki, et al. (2008) J. Virol. 82:5715-24), but thatdepletion of cholesterol ablates particle infectivity (Aizaki, et al.(2008) supra; Kapadia, et al. (2007) J. Virol. 81:374-83).

While clinically approved HCV entry inhibitors have not been identified,a number of agents have been described for inhibiting HCV replication.For example, the current treatment option for HCV is a combinationtherapy with interferon (IFN) and ribavirin. However, this combinationhas toxic side effects, marginal efficacy, and limited availability(Firpi & Nelson (2007) Arch. Med. Res. 38:678-690; Foster & Mathurin(2008) Antivir. Ther. 13:3-8). In addition, US 2008/0161324 describes aseries of HCV replication inhibitors identified using a replicationassay. However, the compounds identified therein were not shown toprevent infection and spread of HCV. As such, the identification ofnovel and more potent antivirals targeting other aspects of the virallife cycles (e.g., entry inhibitors) is imperative.

Ezetimibe, a 2-azetidinone class of drug, is an anti-hyperlipidemic,cholesterol-lowering medication, currently approved for use in humans bythe U.S. Food and Drug Administration (FDA); the drug has been shown topotently inhibit cholesterol absorption in vivo, thus lowering plasmatotal and LDL cholesterol in treated individuals (Bays, et al. (2008)Expert Rev. Cardiovasc. Ther. 6:447-470). Data indicate that the proteinknown as Niemann-Pick C1-like 1 (NPC1L1) is the molecular target ofezetimibe in cells (Garcia-Calvo, et al. (2005) Proc. Natl. Acad. Sci.USA 102:8132-8137). Additional studies indicate that SR-B1 might be thetarget of ezetimibe action in cells (Labonte, et al. (2007) Biochim.Biophys. Acta 1771:1132-1139). Ezetimibe is marketed under the tradenames EZETROL and ZETIA. It is also marketed in combination with thestatin drug simvastatin (ZOCOR) under the trade names VYTORIN and INEGY.It is indicated as an adjunct to dietary measures in the management ofhypercholesterolaemia, homozygous sitosterolemia (phytosterolemia), andthe treatment of mixed hyperlipidaemia when used in combination withfenofibrate.

SUMMARY OF THE INVENTION

The present invention embraces a method for reducing or preventing entryof a hepatic virus into a cell by contacting the cell with an effectiveamount of a Niemann-Pick C1-like 1 (NPC1L1) protein antagonist.According to one embodiment of this method, the NPC1L1 antagonist is anazetidinone-based cholesterol absorption inhibitor. In particularembodiments, the azetidinone-based cholesterol absorption inhibitor isezetimibe, or a derivative thereof.

The present invention also features a method for preventing a hepaticvirus infection by administering to a subject in need of treatment aneffective amount of a NPC1L1 antagonist o that entry of hepatic virusinto cells is reduced or prevented. According to one embodiment of thismethod, the subject has had a liver transplant. In another embodiment,the NPC1L1 antagonist is an azetidinone-based cholesterol absorptioninhibitor. In particular embodiments, the azetidinone-based cholesterolabsorption inhibitor is ezetimibe, or a derivative thereof.

A synergistic composition for treating a hepatic virus infection is alsoembraced by this invention. This synergistic composition is composed ofa NPC1L1 antagonist and at least one other agent that inhibits hepaticvirus infection. NPC1L1 antagonists particularly encompassed includeazetidinone-based cholesterol absorption inhibitors, with particularembodiments drawn to ezetimibe, or a derivative thereof. In certainembodiments, the at least one other agent that inhibits hepatic virusinfection is a type I interferon (IFN). In particular embodiments, thetype I IFN is IFN-α or IFN-β.

Another feature of this invention is a method for effecting clearance ofhepatic virus from cells by contacting cells infected with a hepaticvirus with an effective amount of the synergistic composition of theinvention.

The present invention also embraces a method for treating a hepaticvirus infection. This method involves administering to a subject with ahepatic virus infection an effective amount of the synergisticcomposition of the invention so that the hepatic virus infection istreated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that ezetimibe inhibits HCV internalization, not binding.Monolayers of human hepatoma cell line Huh7 were inoculated with HCVccJFH-1 (cell culture produced HCV JFH-1, which is a specific HCV cloneisolated from a Japanese Fulminant Hepatitis patient) at an MOI of 0.1FFU/cell for 6 hours in the presence or absence of increasingconcentrations of ezetimibe. Cultures were washed extensively and cellassociated RNA was collected 6 and 30 hours post-infection (p.i.) todetermine the amount of bound and internalized virus, respectively. HCVRNA was quantified by RT-qPCR, normalized to GAPDH and is displayed asHCV RNA copies/μg total cellular RNA. Results are graphed asmeans±s.e.m. for triplicate samples. The data presented arerepresentative of three independent experiments. Assay background (i.e.,HCV RNA level detected in uninfected samples) is equal to ˜0.5-1.0×10²copies/μg total cellular RNA. Significant reduction in HCV RNA levelsrelative to mock-treated cultures is denoted by a single asterisk(p<0.05, one-way ANOVA and Tukey's post hoc t test).

FIG. 2 shows that ezetimibe synergizes with IFN-α to potently inhibitHCV RNA levels in chronically infected Huh7 cells. Cultures of growingHuh7 cells were inoculated with HCVcc JFH-1 at an MOI of 0.01 FFU/cell.Infected cultures were maintained for an additional 10 days to allow HCVRNA to reach steady-state levels. FIG. 2A, Cultures were mock treated,treated with ezetimibe alone (30 μM), IFN-α (100 U/ml) alone, orezetimibe (30 μM) plus IFN-α (100 U/ml) in combination. Treatments weremaintained as cultures were passaged. FIG. 2B, After 70 days IFN-α orcombination ezetimibe plus IFN-α treatment, treatment was eithercontinued (open shapes) or discontinued (filled shapes). Throughout theexperiment, intracellular RNA was collected from an aliquot of cellsfrom triplicate cultures on indicated days post-treatment and HCV RNAwas quantified by RT-qPCR, normalized to GAPDH and is displayed as HCVRNA copies/μg total cellular RNA. Results are graphed as means±s.e.m.for triplicate samples. The data presented are representative of twoindependent experiments.

DETAILED DESCRIPTION OF THE INVENTION

It has now been shown that the clinically-available anti-cholesterolmedication ezetimibe, which inhibits cellular cholesterol absorption(Gupta & Ito (2002) Heart Dis. 4:399-409), potently blocks HCV entryinto cells and infection. Unlike statins that act intracellularly tolimit cholesterol metabolism (e.g., by inhibiting the enzyme HMG-CoAreductase) thereby inhibiting HCV RNA replication or particle maturation(Kapadia & Chisari (2005) Proc. Natl. Acad. Sci. USA 102:2561-6; Sagan,et al. (2006) Biochem. Cell. Biol. 84:67-79; Ye, et al. (2003) Proc.Natl. Acad. Sci. USA 100:15865-70; Kapadia & Chisari (2005) DifferentialRegulation of HCV Genotype 1B and 2A RNA Replication by the Cholesteroland Fatty Acid Biosynthetic Pathways. In 12th International Symposium onHepatitis C Virus & Related Viruses (Montreal, Canada)), ezetimibefunctions by preventing cholesterol and HCV particle internalization byantagonizing the Niemann-Pick C1-like 1 (NPC1L1) protein. Moreover, whenused in combination with interferon, ezetimibe synergistically cureschronically-infected cell cultures, reducing HCV RNA and protein toundetectable levels. Because ezetimibe is already approved for otherclinical uses and has been found to be safe for patient use, one ofskill will understand that the present invention could readily translateinto a new, more effective, therapeutic treatment for a hepatic virusinfection. Because it is involved in blocking viral entry into cells(i.e., not just inhibiting virus replication, but preventing viralinfection), ezetimibe and its derivatives can be used in the treatmentof hepatic virus infection (to limit spread within the liver and to anew liver after liver transplantation), as well as in prophylactictreatment of healthcare workers or others at risk of a hepatic virusinfection.

Accordingly, the present invention embraces the use of ezetimibe, aswell as other agents that antagonize NPC1L1 or inhibit cellularcholesterol uptake, in methods for reducing or preventing entry of ahepatic virus into a cell, clearing a hepatic virus infection, andpreventing or treating a hepatic virus infection and spread of virus toa new liver after liver transplantation.

The term “hepatic virus” refers to a virus that can cause viralhepatitis. Viruses that can cause viral hepatitis include hepatitis A,hepatitis B, hepatitis C, hepatitis D, and hepatitis E. In addition,non-ABCDE cases of viral hepatitis have also been reported (see, forexample, Rochling, et al. (1997) Hepatology 25:478-483). Within eachtype of viral hepatitis, several subgroupings have been identified.Hepatitis C, for example, has at least six distinct genotypes (1, 2, 3,4, 5, and 6), which have been further categorized into subtypes (e.g.,1a, 1b, 2a, 2b, 2c, 3a, 4a) (Simmonds (2004) J. Gen. Virol.85:3173-3188). In particular embodiments of the invention, the hepaticvirus is hepatitis C virus (HCV).

An NPC1L1 antagonist is used herein to refer to an agent that reducesthe expression or activity, or inhibits expression or activity, of anNPC1L1 nucleic acid or polypeptide. Examples of antagonists includewithout limitation small molecules, anti-NPC1L1 antibodies, antisensenucleic acids, ribozymes, RNAi oligonucleotides, and molecules thattarget NPC1L1 promoter transcription factors. In particular embodiments,an NPC1L1 antagonist inhibits the activity of NPC1L1 by blockinginternalization or uptake of a hepatic virus into cells. Specific NPC1L1antagonists that inhibit NPC1L1 activity include, for example,azetidinone-based cholesterol absorption inhibitors (e.g., ezetimibe andits derivatives); 4-phenyl-4-piperidinecarbonitrile hydrochloride;1-butyl-N-(2,6-dimethylphenyl)-2 piperidinecarboxamide;1-(1-naphthylmethyl)piperazine;3{1-[(2-methylphenyl)amino]ethylidene}-2,4(3H,5H)-thiophenedione,3{1-[(2-hydroxyphenyl)amino]ethylidene}-2,4(3H,5H)-thiophenedione,2-acetyl-3-[(2-methylphenyl)amino]-2-cyclopenten-1-one,3-[(4-methoxyphenyl)amino]-2-methyl-2-cyclopenten-1-one,3-[(2-methoxyphenyl)amino]-2-methyl-2-cyclopenten-1-one, andN-(4-acetylphenyl)-2-thiophenecarboxamide, or derivatives thereof. See,e.g., US 2009/0035784. In particular embodiments, the NPC1L1 antagonistis an azetidinone-based cholesterol absorption inhibitor.Azetidinone-based cholesterol absorption inhibitors are described, forexample, by Rosenblum, et al. ((1998) J. Med. Chem. 41(6):973-80). Aparticularly preferred azetidinone-based inhibitor for use incompositions and methods of the present invention is ezetimibe(1-(4-fluorophenyl)-(3R)-[3-(4-fluorophenyl)-(3S)-hydroxypropyl]-(45)-(4-hydroxyphenyl)-2-azetidinone)(also referred to in the literature as SCH 58235 or ZETIA) and itsphenolic glucuronide derivative, SCH60663. See van Heek (2000) Br. J.Pharmacol. 129(8):1748-54. Additional ezetimibe related derivatives foruse in compositions and methods of the present invention are referred toin the literature as SCH 58053 or(+)-7-(4-chlorophenyl)-2-(4-flourophenyl)-7-hydroxy-3R-(4-hydroxyphenyl)-2-azaspiro[3,5]nonan-1-one)(Repa, et al. (2002) J. Lipid Res. 43:1864-1874); and SCH 48461 or(3R)-3 Phenylpropyl)-1,(4S)-bis(4-methoxyphenyl)-2-azetidinone(Salisbury, et al. (1995) Atherosclerosis 115:45-63). Moreover, NPC1L1antagonists, e.g., small molecule antagonists, can be identified usingconventional screening assays, which monitor cholesterol absorption orassays which monitor hepatic virus entry into a cell as describedherein. Whether used alone or in combination with one or more agents,desirably the NPC1L1 antagonist is used in the range including, but notlimited to, 10 to 50 μM.

According to one embodiment of the invention, an NPC1L1 antagonist isused in a method to reduce or prevent entry of a hepatic virus into acell. This method involves contacting a cell with an NPC1L1 antagonistso that entry of a hepatic virus into a cell is reduced or prevented. Inthis respect an effective amount is an NPC1L1 antagonist is an amountwhich results in a 20% to 100% decrease in internalization, entry oruptake of a hepatic virus into a cell as compared to a cell notcontacted with the NPC1L1 antagonist. Such a decrease in internalizationcan be determined using the techniques disclosed herein or any othersuitable method for monitoring virus internalization, e.g., enzymaticreporter assays or monitoring intracellular localization by confocalmicroscopy. In particular embodiments of the present invention, the cellis a hepatic cell. In so far as entry of a hepatic virus into a cell isessential for establishing a hepatic viral infection, this method findsapplication in the preventing a hepatic virus infection in a subject.

Accordingly, another embodiment of the present invention embraces amethod for preventing hepatic virus infection by administering to asubject in need of treatment an effective amount of a NPC1L1 antagonistso that entry of a hepatic virus into cells is reduced or preventedthereby preventing a hepatic virus infection. By “subject” is meant anyanimal (e.g., a mammal such as a human). As used herein, the term“hepatic virus infection” is used to describe the process of adherenceand internalization of a hepatic virus, which is manifested by viralreplication and viral persistence. Thus, “prevention” or “preventing” inthe context of the present invention refers to prophylactic treatmentwhich prevents or delays HCV-associated clinical symptoms. In thisrespect, subjects benefiting from prophylactic treatment with a NPC1L1antagonist include, e.g., healthcare workers or others at risk of ahepatic virus infection, as well as hepatic virus-positive livertransplant patients, wherein prophylactic treatment prevents infectionof the new liver.

In so far as the activity of ezetimibe was shown to synergize with theactivity of interferons, the present invention also embraces asynergistic composition that includes a NPC1L1 antagonist in combinationwith at least one additional agent that inhibits hepatic virus infection(e.g., by inhibiting gene expression, replication, assembly, maturation,or release) for use in the treatment of a hepatic virus infection. Acomposition of the invention is deemed synergistic since the individualcomponents, when combined, have a total effect that is greater than thesum of the individual effects. Suitable NPC1L1 antagonists are disclosedherein, with particular embodiments embracing azetidinone-basedcholesterol absorption inhibitors such as ezetimibe or its derivatives.In this respect, NPC1L1 antagonists, as hepatic virus entry inhibitors,are distinct from replication inhibitors. Thus, according to oneembodiment of the invention, the at least one additional agent thatinhibits hepatic virus infection is an inhibitor of viral replication.Agents that inhibit hepatic virus replication are known in the art andcan target a variety of different replication mechanisms. For example,HCV replication can be inhibited by reducing the rate of any of thesteps required for its replication or inhibiting any molecule involvedin replication, including but not limited to, viral genome replication,translation of viral RNA, and protolytic processing. Agents of use inthe compositions and methods of the invention include, but are notlimited to, iminothiazolidinones (U.S. Pat. No. 7,183,302), type Iinterferons (Zeuzem, et al. (1996) Hepatology 23(2):366-71), ribavirin(Gish (2006) J Antimicrob Chemother. 57(1):8-13), nucleoside analogR1479 (Klumpp, et al. (2006) J. Biol. Chem. 281(7):3793-9), Telaprevir(Weisberg & Jacobson (2009) Clin. Liver Dis. 13(3):441-52; Serrazin, etal. (2007) Gastroenterology 132(5):1767-77), Boceprevir (Mederacke, etal. (2009) Curr. Opin. Investig. Drugs 10(2):181-9), nucleosideinhibitor R1626 (Toniutto, et al. (2008) IDrugs 11(10):738-49), ITMN-191(R7227; Seiwert, et al. (2008) Antimicrob. Agents Chemother.52(12):4432-41), substituted diphenyl heterocyclic compounds (Huang, etal. (2008) Antimicrob. Agents Chemother. 52(4):1419-29),beta-D-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130; Asif, et al.(2007) Antimicrob. Agents Chemother. 51(8):2877-82), statins (Ikeda, etal. (2006) Hepatology 44(1):117-25), bisindolylmaleimides andindolocarbazoles (Murakami, et al. (2009) Antiviral Res. 83:112-117),and combinations thereof. In particular embodiments, the synergisticcomposition does not include simvastatin.

In particular embodiments, the synergistic composition of the inventionincludes at least a type I interferon (e.g., IFN-α or IFN-β), ribavirin,or a combination thereof. In this respect, one embodiment embraces asynergistic composition composed of 1) a NPC1L1 antagonist, 2) a type Iinterferon or ribavirin, 3) and an additional agent that inhibitshepatic virus infection. As used herein, a type I interferon refers tothe family of interferon proteins that inhibit viral replication,inhibit cellular proliferation, and modulate immune response. There area variety of commercially available alpha interferons, including, butnot limited to, Roferon A interferon (Hoffman-La Roche, Nutley, N.J.),Berofor alpha 2 (Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield,Conn.), and Sumiferon (Sumitomo, Japan). Alpha interferon 2b currentlyhas the broadest approval throughout the world for use in treating HCV.U.S. Pat. No. 4,530,901 provides a description of the manufacture ofalpha interferon 2b.

For use in the methods of the invention, desirably the synergisticcomposition is formulated for administration to a subject. In thisrespect, the NPC1L1 antagonist and at least one additional agent thatinhibits hepatic virus infection can be combined in appropriate amountsin admixture with one or more pharmaceutically acceptable carriers. Suchcarriers are well-known in the art and include, e.g., saline solution,cellulose, lactose, sucrose, mannitol, sorbitol, and calcium phosphates.Optional additives include lubricants and flow conditioners, e.g.,silicic acid, silicon dioxide, talc, stearic acid, magnesium/calciumstearates and polyethylene glycol (PEG) diluents; disintegrating agents,e.g., starch, carboxymethyl starch, cross-linked PVP, agar, alginic acidand alginates, coloring agents, flavoring agents and melting agents.Dyes or pigments may be added to tablets or dragee coatings, forexample, for identification purposes or to indicate different doses ofactive ingredient.

Generally, the active ingredients are present in an amount of 1-95% byweight of the total weight of the synergistic composition. Thecomposition may be provided in a dosage form that is suitable for theoral, parenteral (e.g., intravenously or intramuscularly), rectal,determatological, cutaneous, nasal, vaginal, inhalant, skin (patch), orocular administration route. Thus, the composition may be in the formof, e.g., tablets, capsules, pills, powders, granulates, suspensions,emulsions, solutions, gels including hydrogels, pastes, ointments,creams, plasters, drenches, osmotic delivery devices, suppositories,injectables, implants, sprays, or aerosols. The pharmaceuticalcompositions may be formulated according to conventional pharmaceuticalpractice (see, e.g., Remington:The Science and Practice of Pharmacy,20th edition, 2000, ed. A. R. Gennaro, Lippincott Williams & Wilkins,Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J.Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

It is contemplated that the NPC1L1 antagonist and at least oneadditional agent that inhibits hepatic virus infection can be formulatedin a single formulation or multiple formulations with simultaneous orconsecutive (e.g., within minutes, days or hours) administration.

The dosage of a compound or a combination of compounds depends onseveral factors, including the administration method, the type of viralhepatitis to be treated, the severity of the infection, whether dosageis designed to treat or prevent a viral hepatitis infection, and theage, weight, and health of the patient to be treated.

Because the synergistic composition of the invention targets both viralentry and gene expression, replication, assembly, maturation and/oregress, this composition finds use in methods for effecting clearance ofa hepatic virus from a cell and treating a hepatic virus infection.Accordingly, the present invention also embraces a method for effectingclearance of a hepatic virus from a cell. This method involvescontacting a hepatic virus-infected cell with an effective amount of thesynergistic composition of the invention so that the hepatic virus iscleared from the cell. Desirably, the synergistic composition providesat least a 20%, 30%, 50%, 70%, 80%, 90%, 95%, or 99% decrease in viralload from the cell, as determined using a suitable assay. According toparticular embodiments of this invention, a cell of this method is ahepatic cell. By affecting clearance of a hepatic virus from a cell,this method of the invention finds use in the treatment of a hepaticvirus infection.

Accordingly, another embodiment of the present invention embraces amethod for treating a hepatic virus infection by administering to asubject with a hepatic virus infection an effective amount of thesynergistic composition of the invention so that the hepatic virusinfection is treated. In the context of the present invention, “treat”or “treating” refers to the administration of a synergistic compositionto measurably slow or stop viral replication or spread, to measurablydecrease the load of a virus (e.g., any virus described herein includinga hepatitis virus such as hepatitis A, B, C, D, or E), and/or to reduceat least one symptom associated with a hepatic virus infection.Desirably, the slowing in replication or the decrease in viral load isat least 20%, 30%, 50%, 70%, 80%, 90%, 95%, or 99%, as determined usinga suitable assay (e.g., a replication assay or infection assay describedherein).

Subjects benefiting from treatment include those diagnosed with ahepatic virus infection, e.g., an individual in which a hepatic virusmarker has been detected. A variety of markers are known in the art andcan be readily measured by a skilled artisan. For example, HCV infectioncan be diagnosed by the presence of the viral genome or proteins in theliver or blood.

In the context of prophylactic and therapeutic use, “an effectiveamount” of a composition herein is defined as an amount of an agentwhich reduces or eliminates viral load to reduce, mitigate or eliminagechronic infection, which leads to cancer or cirrhosis (with symptomssuch as enlarged liver, enlarged spleen, jaundice, muscle wastingexcoriations, ascites and ankle swelling).

An effective amount for use in accordance with the present methods canbe determined by a variety of means well known to those of skill in theart. For example, it is contemplated that one of skill in the art canchoose an effective amount using an appropriate animal model system,e.g., as described herein, to test for inhibition of HCV in vivo. Themedical literature provides detailed disclosure on the advantages anduses of a wide variety of such models. Once a test drug has shown to beeffective in vivo in animals, clinical studies can be designed based onthe doses shown to be safe and effective in animals. One of skill in theart can design such clinical studies using standard protocols asdescribed in textbooks such as Spilker ((2000) Guide to Clinical Trials.Lippincott Williams & Wilkins:Philadelphia).

In the present invention, doses of drugs to be administered in themethods of the present invention can be chosen by one of skill in theart based on the known pharmacology and toxicology of ezetimibe, or itsderivatives, and interferon or other inhibitors as they are used inclinical medicine. One of skill would choose doses using resources suchas the currently approved product labeling for these drugs. It iscontemplated that these doses would be individualized for a patientbased on the judgment of the physician.

If desired, the compounds of the invention may be employed inmechanistic assays to determine whether other combinations, or singleagents, are as effective as the combinations of the invention ininhibiting a viral disease using assays generally known in the art. Forexample, candidate compounds can be tested, alone or in combination(e.g., with a NPC1L1 antagonist) and applied to cells (e.g., hepaticcells such as Huh7, Huh2, Huh 8, Sk-Hep-1, Huh7 lunet, HepG2, WRL-68,FCA-1, LX-1, LX-2, Huh7-derived cells lines). After a suitable time,viral entry and replication or load of these cells is examined. Adecrease in viral entry (e.g., as determined by a standard fociformation assay), replication or viral load identifies a candidatecompound or combination of agents as an effective agent for treating aviral disease.

The compounds disclosed herein are also useful tools in elucidatingmechanistic information about the biological pathways involved in viraldiseases. Such information can lead to the development of newcombinations or single agents for treating, preventing, or reducing aviral disease. Methods known in the art to determine biological pathwayscan be used to determine the pathway, or network of pathways affected bycontacting cells (e.g., hepatic cells) infected with a virus with thecompounds of the invention. Such methods can include, analyzing cellularconstituents that are expressed or repressed after contact with thecompounds of the invention as compared to untreated, positive ornegative control compounds, and/or new single agents and combinations,or analyzing some other activity of the cell or virus such as anenzymatic activity, nutrient uptake, and proliferation. Cellularcomponents analyzed can include gene transcripts, and proteinexpression. Suitable methods can include standard biochemistrytechniques, radiolabeling the compounds of the invention, and observingthe compounds binding to proteins, e.g., using 2D gels and/or geneexpression profiling. Once identified, such compounds can be used in invivo models (e.g., knockout or transgenic mice) to further validate thetool or develop new agents or strategies to treat viral disease.

The invention is described in greater detail by the followingnon-limiting examples.

EXAMPLE 1 Methods

Cells. Huh7 cells, also known as Huh7/scr cells, are well-known in theart (Zhong, et al. (2005) Proc. Natl. Acad. Sci. USA 102, 9294-99;Gastaminza, et al. (2006) J. Virol. 80:11074-81; Zhong, et al. (2006) J.Virol. 80:11082-93; Sainz et al. (2009) PLoS ONE 4:e6561). The Clone BHCV genotype 1b sub-genomic replicon (sg1b) Huh7 cells were obtainedthrough the NIH AIDS Research and Reference Reagent Program and havebeen previously described (Blight, et al. (2000) Science 290:1972-1974).The HCV sg2a replicon was established as previously described(Uprichard, et al. (2006) Virol. J. 3:89). Briefly the HCV genotype 2asubgenomic replicon RNA (psgJFH1; Kato, et al. (2003) Gastroenterology125:1808-17) was XbaI linearized, in vitro transcribed using MEGASCRIPTT7 (Ambion, Austin, Tex.) and transfected into cells using a modifiedelectroporation protocol (Krieger, et al. (2001) J. Virol.75(10):4614-24). All cells were cultured in complete Dulbecco's modifiedEagle's medium (cDMEM) (Hyclone, Logan, Utah) supplemented with 10%fetal bovine serum (FBS) (Hyclone), 100 units/ml penicillin, 100 mg/mlstreptomycin, and 2 mM L-glutamine (Gibco Invitrogen, Carlsbad, Calif.)and 500 μg/ml geneticin ((Invitrogen) for HCV replicon cells only).

HCVcc Generation. The plasmid containing the full-length JFH-1 genome(pJFH1) has been previously described (Kato, et al. (2003) supra; Kato,et al. (2001) J. Med. Virol. 64:334-9; Wakita, et al. (2005) Nat. Med.11:791-6). Protocols for JFH-1 in vitro transcription and HCV RNAelectroporation have been described (Uprichard, et al. (2006) supra).The JFH-1 HCVcc viral stock was generated by infection of naive Huh7cells at a multiplicity of infection (MOI) of 0.01 focus forming units(FFU)/cell, using medium collected of day 18 post-electroporation ofHuh7 cells with in vitro transcribed JFH-1 RNA (Zhong, et al. (2005)supra).

Reagents. Recombinant human interferon-a 2a (IFN-α2a) and IFN-β (PBLBiomedical Laboratories, New Brunswick, N.J.) were resuspended to aconcentration of 50 U/μl in complete DMEM supplemented with 10% FBS,aliquoted into single use tubes, and stored at −80° C. Ezetimibe wasresuspended to a concentration of 20 mM in DMSO and stored at 4° C.

Treatment of Acute HCVcc Infections. For experiments in growing cells,Huh7 cells were seeded 24 hours prior to use at 4×10³ cells in each wellof a 96-well plate (BD Biosciences, San Jose, Calif.). For experimentsin non-growing cells, Huh7 cells were seeded in 96-well BIOCOAT cultureplates (BD Biosciences) at a density of 8×10³ cells/well in cDMEM. Uponreaching 90% confluence, media was replaced with 200 μl cDMEMsupplemented with 1% (v/v) DMSO (Sigma), and cells were cultured for anadditional 20 days, replacing medium every 2 days (Sainz, Jr. & Chisari(2006) J. Virol. 80:10253-7; Choi, et al. (2009) Xenobiotica 39:205-17).Cells were infected with HCVcc JFH-1 at an MOI of 1.0 or 0.1 FFU/cellfor 12 hours. Cells were mock-treated or treated with increasingconcentrations of ezetimibe 6 hours prior to infection (PRE), during thetime of infection for 12 hours (CO), or immediately following infectionfor 60 hours (POST). For RT-qPCR analysis, total cellular RNA wasextracted in 1× Nucleic Acid Purification Lysis Solution (AppliedBiosystems, Foster City, Calif.) from triplicate wells at indicatedtimes post infection. For HCV E2-positive foci assay analysis, mediumwas removed and cells were fixed with 4% paraformaldehyde (w/v) (Sigma)72 hours post-infection and immunohistochemical staining for HCV E2 wasperformed as described herein.

Treatment of Chronic HCVcc Infections. For experiments in growing cells,Huh7 cells seeded at 1×10⁶ cells in a T75 cell culture flask (Corning)were infected with JFH-1 HCVcc at a MOI of 0.01 FFU/cell andsubsequently cultured for 10 days to allow HCV RNA to reach steady-statelevels. On day 10 post-infection, cells were split 1:4 and re-platedinto T25 culture flasks (Corning). Twenty four hours post seeding,individual flasks were mock-treated with cDMEM or treated with cDMEMcontaining IFN-α (100 U/ml), ezetimibe (30 μM) or a combination of bothIFN-α (100 U/ml) and ezetimibe (30 μM). Throughout the course of theexperiment, fresh media with treatments was replenished every two daysand cells were trypsinized just before reaching confluence and re-platedat a dilution of 1:3 to maintain active cell growth. Samples werecollected at the time of each splitting, excess cells were pelleted at1200 rotations per minute for 5 minutes and total RNA was isolated in 1×Nucleic Acid Purification Lysis Solution (Applied Biosystems) forreverse transcription followed by RT-qPCR analysis.

For experiments in non-growing cells, Huh7 cells were seeded in 48-wellBIOCOAT culture plates (BD Biosciences) at a density of 1×10⁴ cells/welland subsequently cultured in the presence of 1% DMSO for 20 days asdescribed herein. Cultures were infected with JFH-1 HCVcc at a MOI of0.01 FFU/cell and maintained for 14 days to allow HCV RNA to reachsteady-state levels. On day 14 post-infection, wells were mock-treatedwith cDMEM or treated with DMEM containing IFN-α, ezetimibe or acombination of both, as described herein for growing Huh7 cultures.Media and respective treatments were replenished every 2 days. Onindicated days post-treatment, total RNA was isolated from triplicatewells in 1× Nucleic Acid Purification Lysis Solution (AppliedBiosystems) for reverse transcription followed by RT-qPCR analysis.

RNA Isolation and RT-qPCR Analysis. Total cellular RNA was purifiedusing an ABI PRISM™ 6100 Nucleic Acid PrepStation (Applied Biosystems),as per the manufacturer's instructions. One Ag of purified RNA was usedfor cDNA synthesis using the TAQMAN reverse transcription reagents(Applied Biosystems), followed by SYBR green RT-qPCR using an AppliedBiosystems 7300 real-time thermocycler (Applied Biosystems). Thermalcycling included of an initial 10-minute denaturation step at 95° C.followed by 40 cycles of denaturation (15 seconds at 95° C.) andannealing/extension (1 minute at 60° C.). HCV JFH-1 and GAPDH transcriptlevels were determined relative to a standard curve derived from serialdilutions of plasmid containing the JFH-1 HCV cDNA or the human GAPDHgene, respectively. The PCR primers used to detect GAPDH and HCVwere:human GAPDH (NMX002046) 5′-GAA GGT GAA GGT CGG AGT C-3′ (sense; SEQID NO:1) and 5′-GAA GAT GGT GAT GGG ATT TC-3′ (anti-sense; SEQ ID NO:2);and JFH-1 HCV (AB047639) 5′-TCT GCG GAA CCG GTG AGT A-3′ (sense; SEQ IDNO:3)) and 5′-TCA GGC AGT ACC ACA AGG C-3′ (anti-sense; SEQ ID NO:4).

Extracellular Infectivity Titration Assay. Cell supernatants wereserially diluted 10-fold in cDMEM and 100 μl was used to infect, intriplicate, 4×10³ naive Huh7 cells per well in 96-well plates (BDBiosciences). The inoculum was incubated with cells for 24 hours at 37°C. and then overlayed with 150 μl complete DMEM containing 0.4%methylcellulose (w/v) (Fluka BioChemika, Switzerland) to give a finalconcentration of 0.25% methylcellulose. Seventy-two hourspost-infection, medium was removed, cells were fixed with 4%paraformaldehyde (Sigma) and immunohistochemical staining for HCV E2 wasperformed. Briefly, cells were first incubated with 1×PBS containing0.3% (v/v) hydrogen peroxide (Fisher, Fairlawn, N.J.) to blockendogenous peroxidase. Following three rinses with 1×PBS, cells wereblocked for 1 hour with 1×PBS containing 0.5% (v/v) TRITON X-100(Fisher), 3% (w/v) bovine serum albumin (BSA) (Sigma) and 10% (v/v) FBS.The HCV E2 glycoprotein was detected by incubation at room temperaturewith 1×PBS containing 0.5% (v/v) TRITON X-100 and 3% (w/v) BSA and a1:500 dilution of the human monoclonal anti-HCV E2 antibody C1. Bound C1was subsequently detected by a 1 hour incubation with a 1:1000 dilutionof an HRP-conjugated anti-human antibody (Pierce, Rockford, Ill.)followed by a 30 minute incubation with an AEC detection substrate (BDBiosciences). Cells were washed with distilled H₂O and visualized usinga ZEISS AXIOVERT microscope (Carl Zeiss, Germany). Viral infectivitytiters are expressed as FFU per milliliter of supernatant (FFU/ml),determined by the average number of E2-positive foci detected intriplicate samples at the highest HCV-positive dilution.

Western Blot Analysis. Cells were harvested in 1.25% TRITON X-100 lysisbuffer (TRITON X-100, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA)supplemented with a protease inhibitor cocktail (Roche Applied Science,Indianapolis). Fifty micrograms of protein was resolved by SDS-PAGE andtransferred to HYBOND nitrocellulose membranes (Amersham Pharmacia,Piscataway, N.J.). Membranes were sequentially blocked with 5% NonfatMilk, incubated with a 1:1000 dilution of a monoclonal mouse anti-HCVNS3 antibody (Clone 9-G2, ViroGen, Watertown, Mass.), washed 3 timeswith PBS/0.05% TWEEN 20, incubated with horseradishperoxidase-conjugated goat anti-mouse antibody (Pierce, Rockford, Ill.),and washed again. Bound antibody complexes were detected withSUPERSIGNAL chemiluminescent substrate (Pierce).

Cell Proliferation and Cytotoxicity Bioluminescence Assays. The VIALIGHTPlus Cell Proliferation assay kit (Lonza, Walkersville, Md.), whichincorporates bioluminescent detection of cellular ATP as a measure ofcell viability and proliferation, was used according to themanufacturer's instructions. Briefly, mock-treated and drug-treatedcultures were lysed in Cell Lysis reagent for minutes. One hundred μl ofculture medium was transferred to white 96-well plates (BD Biosciences)containing ATP detection reagent and luminescence, expressed as relativelight units (RLU), was measured (Fluostar OPTIMA). To assessdrug-induced cellular toxicity, a bioluminescence-based assay (TheTOXILIGHT BioAssay Kit, Lonza) that measures adenylate kinase (AK)released from damaged cells was used as per the manufacturer'sinstructions. Briefly, 20 μl of supernatant was collected on indicateddays and transferred to white 96-well plates (BD Biosciences). Onehundred μl of AK detection reagent was then added to each well, andluminescence (RLU) was measured (Fluostar OPTIMA). In general,cytotoxicity analysis demonstrated that ezetimibe treatment of Huh7cells for 12 hours was not cytotoxic at the concentrations tested.

Statistics. Data are presented as the means±standard error of the means(sem). Significant differences were determined by one-way analysis ofvariance (ANOVA) followed by Tukey's post hoc t test (GRAPHPAD PRISMSoftware, San Diego, Calif.).

EXAMPLE 2 Ezetimibe Blocks Entry of HCV Into Cells

Ezetimibe, a 2-azetidinone class of drug, is an anti-hyperlipidemic,cholesterol-lowering medication which has been shown to inhibitcholesterol absorption in vivo, lowering plasma LDL and totalcholesterol in treated individuals (Gupta & Ito (2002) Heart Dis.4:399-409; Bays, et al. (2008) Expert Rev. Cardiovasc. Ther. 6:447-70).Since infectious HCV particles are enriched in cellular cholesterol(Aizaki, et al. (2008) J. Virol. 82:5715-24), it was contemplated thatezetimibe might also inhibit HCV entry into cells. As such, the abilityof ezetimibe to inhibit HCV entry was evaluated by performing a HCV focireduction assay. Huh7 cells were inoculated with cell culture-producedHCV (HCVcc) at a multiplicity of infection (MOI) of 1.0 or 0.1 focusforming units (FFU)/cell and treated with increasing concentrations (0,3.125, 6.25, 12.5 and 25 μM) of ezetimibe pre-, co- or post-infection.Ezetimibe reduced HCV foci formation in a dose-dependent manner relativeto untreated cells (0 μM ezetimibe) when present for 6 hours prior toinfection and then removed or during the 12 hour virus inoculationperiod and then removed. Specifically, pre-treatment of cells with 12.5or 25 μM ezetimibe, inhibited HCV foci formation by 85 and 95%,respectively, compared to untreated cultures, while more potentinhibition of 90 and 99% was observed when cells were co-incubated withHCVcc and ezetimibe at 12.5 μM or 25 μM, respectively. However, thedegree of inhibition observed was greatly reduced when ezetimibe wasadded to cells post-infection (p.i.).

Likewise dose-dependent and time-of-addition-dependent inhibition of HCVinfection was observed when HCV RNA levels were quantified by RT-qPCR24, 48 and 72 hours p.i. Notably, pre-treatment with 25 μM ezetimibe for6 hours before infection appeared to protect cells from subsequentinfection for at least 48 hours. Co-treatment also potently inhibitedHCV infection, but again consistent with the effects of an entryinhibitor, addition of ezetimibe post-infection was ineffective withonly marginal reduction in HCV RNA levels detected at 72 hours p.i.

To ensure that the inhibition observed was not due to ezetimibe-inducedchanges in cell proliferation or cytotoxicity, cellular ATP was measuredas a read-out of Huh7 cell viability and proliferation and cellulartoxicity by adenylate kinase release following treatment with increasingdoses of ezetimibe. These results confirmed that ezetimibe treatment didnot inhibit cell proliferation or induce cellular toxicity at any of thedoses used. Furthermore, similar ezetimibe-mediated inhibition wasobtained in analogous experiments performed using non-growing Huh7 cellcultures (Sainz, Jr. & Chisari (2006) J. Virol. 80:10253-7), whichbetter mimic the non-dividing state of hepatocytes in vivo. Furthermore,to test the specificity of the observed HCV inhibition, the effect ofezetimibe on entry of another flavivirus, Dengue virus (DNV), wasassessed. Unlike HCV, no significant reduction in DNV plaque formationwas observed upon treating cells with 12.5 or 25 μM ezetimibe. Thustaken together, these data indicate that ezetimibe is a potent andspecific inhibitor of an early step in HCVcc infection.

To confirm that ezetimibe does not inhibit HCV RNA replication, as hasbeen shown for other anti-hyperlipidemic cholesterol-loweringmedications that target the cholesterol biosynthetic pathway rather thanuptake of free cholesterol, the effect of ezetimibe on HCV replicationwas directly assayed by treating Huh7 cells constitutively replicatingHCV genotype 2a or 1b subgenomic (sg) replicons with increasing doses ofezetimibe. As a positive control, cells were also treated in paralleltreatment with Lovastatin, an inhibitor of the cholesterol biosyntheticpathway that has been shown to reduce HCV sglb RNA replication (Kapadia& Chisari (2005) Proc. Natl. Acad. Sci. USA 102:2561-6; Ye, et al.(2003) Proc. Natl. Acad. Sci. USA 100:15865-70; Tobert (2003) Nat. Rev.Drug Discov. 2:517-26) and HCVcc particle secretion (Kapadia & Chisari(2005) supra). Consistent with previous reports, Lovastatin reduced sglbRNA levels 22-fold by 72 hours post-treatment, as determined by RT-qPCRanalysis, but had no affect on sg2a RNA levels. In contrast, nosignificant reduction of sg1b or sg2a HCV RNA steady-state levels wasobserved in the presence of increasing doses of ezetimibe. Similarresults were obtained at 24 and 48 hours post-treatment. Additionally,when evaluated as an inhibitor of HCVcc foci formation, Lovastatin hadno affect when added pre-, co- or post-infection. Thus, together, theseresults show that while inhibitors of cholesterol biosynthesis, such asLovastatin, inhibit HCV RNA replication and particle egress, unlikeezetimibe they do not affect HCV entry.

To decipher whether ezetimibe inhibits HCV cellular binding orsubsequent internalization, cell-associated HCV RNA and protein in mock-and ezetimibe-treated HCVcc-infected cultures was examined. Consistentwith ezetimibe not inhibiting virion binding, similar levels of HCV RNAwere detected in inoculated cells 6 hours p.i, in the absence orpresence of ezetimibe (FIG. 1). In contrast, HCV entry, indicated by HCVRNA expansion and de novo NS5A protein expression was not observed atlater time points in ezetimibe-treated cultures, indicating thatalthough HCV can efficiently bind to ezetimibe-treated Huh7 cells, apost-binding step required for infection initiation was prevented.

Importantly, because IFN-α has been the foundation of chronic HCVtreatment since the 1980′s, experiments examining the combined effect ofinterferon with ezetimibe were performed on chronically-infected Huh7cultures. Specifically, Huh7 cells were infected with HCVcc and culturedfor 14 days to allow HCV RNA to reach steady-state levels. Parallelcultures were then mock-treated or treated with ezetimibe, IFN-α or acombination of both while being maintained in an actively growing statefor −70 days by splitting cultures 1:3 upon reaching 90% confluence.Consistent with an entry inhibitor, addition of ezetimibe alone did notreduce intracellular HCV steady-state RNA levels throughout the courseof treatment, while IFN-α (100 U/ml) alone decreased intracellular HCVRNA levels by ˜2 logs by day 32 post-treatment compared to themock-treated culture. Notably, when IFN-α was used in combination with15 μM or 30 μM ezetimibe (FIG. 2A), a strong synergistic affect wasobserved. IFN-α in combination with 30 μM ezetimibe potently reduced HCVRNA to background levels (≧5 logs decrease) by 32 days post-treatment,indicating that the chronically-infected culture had been cured.

To confirm HCV clearance, drug treatments were subsequently discontinuedon day 70 and HCV RNA levels were monitored for an additional 30 days.While an immediate rebound in HCV RNA to levels equivalent to thosemeasured in non-treated control cultures was observed in cultures thathad been previously maintained in the presence of 100 U/ml IFN-α (FIG.2B), no rebound in HCV RNA (FIG. 2B) or evidence of HCV proteins byimmunohistochemical staining was observed in cultures that had beenpreviously maintained in the presence of combination treatment withIFN-α (100 U/ml) and ezetimibe (30 μM). IFN-α also acted synergisticallywith ezetimibe in non-growing chronically infected Huh7 cell culturesresulting in over a log greater reduction in HCV RNA by day 56 posttreatment compared to IFN-α treatment alone.

Similar studies were carried out with IFN-β alone (20 U/ml), orezetimibe and IFN-β in combination (a combination of either 15 or 30 μMezetimibe with 20 U/ml IFN-β) Intracellular RNA was collected fromtriplicate wells weekly for 8 weeks post-treatment and HCV RNA wasquantified by RT-qPCR. Ezetimibe treatment alone did not reduce HCVinfection (i.e., steady-state intracellular HCV RNA levels) during theexperiment. IFN-β treatment alone significantly reduced intracellularHCV RNA levels (approximately 100-fold) from week 3 to week 8 ascompared to mock treated (controls), indicating pharmacological activityto inhibit chronic HCV infection. Importantly, however, a synergisticeffect was observed when IFN-β was used in combination with ezetimibe atconcentrations of 15 or 30 μM. Specifically, IFN-β (20 U/ml) incombination with 30 μM ezetimibe synergistically reduced HCV RNA levelsby >10,000-fold by week 4 post treatment and by >50,000-fold by week 6to week 8 post treatment, as compared to mock treated HCV cultures(controls). The level of HCV RNA copies measured were equivalent tobackground levels, indicating that the cultures had been “cured” of HCV.It was confirmed that the inhibition observed was not a consequence oftreatment-induced cytotoxicity.

Together, these data demonstrate that regardless of the degree of hosthepatocyte proliferation, ezetimibe functions in synergy with both IFN-αand IFN-β to potently reduce chronic HCV infection in vitro. Therefore,whether alone or as an adjunct therapy to current HCV interferon therapyprotocols, ezetimibe finds application in the prevention or treatment ofHCV infection.

EXAMPLE 3 Role of NPC1L1 in HCV Entry

Studies have shown that a G451R mutation in the viral envelopeglycoprotein (E2) results in HCV virions of higher density and fasterinfection kinetics (Zhong, et al. (2006) J. Virol. 80:11082-11093). Inaddition it has been determined that this mutant displays a reduceddependence on SR-B1 for cellular entry (Grove, et al. (2008) J. Virol.82:12020-12029). Thus, if ezetimibe was blocking HCV entry via an SR-B1mediated mechanism, it would be expected that the mutant G451R viruswould be less susceptible to the effects of ezetimibe. As a result,experiments were performed to assess the antiviral efficacy of ezetimibeagainst the SR-B1-independent HCVcc^(G451R) virus. Monolayers of Huh7cells were inoculated with either wild-type HCVcc^(JFH-1) orHCVcc^(G451R) at an MOI of 0.01 FFU/cell for 12 hours. Ezetimibetreatment at concentrations of 15 or 30 μM was initiated (and thencontinued) either 6 hours prior to infection, during the 12 hourincubation of the virus on the cells, or immediately following the 12hour viral inoculation period. Ezetimibe at a concentration of 30 μMinhibited HCVcc^(JFH-1) infection approximately 10-fold at 48 hourspost-infection. The HCVcc^(G451R) virus was significantly more sensitiveto the inhibitory effects of ezetimibe with virtually 100% of theinfection being blocked by ezetimibe, as indicated by the lack ofsignificant intracellular HCV RNA following infection. These dataindicate that the inhibitory effect of ezetimibe on HCV entry is notmediated by SR-B1.

Therefore, it was determined whether the Neiman-Pick C1 Like-1cholesterol uptake receptor (Ge, et al. (2008) Cell Metab. 7:508-19;Chang & Chang (2008) Cell Metab. 7:469-71; Weinglass, et al. (2008)Proc. Natl. Acad. Sci. USA 105:11140-5) plays a direct role as a HCVentry receptor.

NPCIL1 Silencing Inhibits HCVcc Infectivity. To more directly confirmthat NPC1L1 is an HCVcc entry factor, it was determined whethersilencing of human NPC1L1 by RNA interference (RNAi) would reduce HCVccinfection in Huh7 cells. In addition, a siRNA specific for the known HCVreceptor, scavenger receptor class B member I (SR-BI), was included as apositive control. Huh7 cells were transfected with either control siRNAsor siRNAs specific for human NPC1L1 or SR-B1 and knockdown was confirmedby RT-qPCR. Cells with NCPIL1 or SR-B1 knockdown were subsequentlyinfected with HCVcc 48 hours post-transfection. Consistent with beingHCV entry factors, both SR-B1 and NPC1L1 knockdown significantly reducedHCVcc infectivity (>90%) independently confirming that NPC1L1 isnecessary for HCVcc infection.

HCVcc Binds to NPC1L1 Expressing CHO Cells. To determine if HCVccparticles bind NPC1L1 in the absence of the other HCV receptors, ChineseHamster Ovary Cells (CHO cells) were transiently transfected with anNPC1L1 expression vector (pCDNA3.1-huNPC1L1-HA) and incubated with HCVccfor 6 hours. Following rigorous washing, bound virus was measured byRT-qPCR analysis. The results of this analysis indicated that CHO cellsexpressing SR-B1 but not CD81 bound HCVcc. Notably, similar to CHO cellsexpressing SR-B1, CHO cells expressing only NPC1L1 bound 3 times moreHCVcc than CHO expressing a GFP or empty vector control plasmid.Together, these data indicate that NPC1L1 is an HCV entry factor andthat HCVcc directly interacts with this receptor. As such, the data alsoindicate that other compounds that block NPC1L1 are also useful asinhibitors of HCV entry.

EXAMPLE 4 In Vivo Activity of Ezetimibe

Studies of HCV infection and agents that can be used to prevent or treatinfection have been hampered by the lack of small animal models of HCVinfection. Although efforts to develop small animal infection modelshave included use of tree shrews (Xie, et al. (1998) Virology244:513-520; Zhao, et al. (2002) J. Clin. Invest. 109:221-232),marmosets/tamarins (Feinstone, et al. (1982) J. Infect. Dis.144:588-598; Karayiannis, et al. (1983) J. Med. Virol. 11:251-256;Watanabe, et al. (1987) J. Med. Virol. 22:143-156), and other primates(Abe, et al. (1993) J. Med. Primatol. 22:433-434), successfultransmission of HCV infection has only been observed in the chimpanzee.Recently, hepatic xenorepopulation approaches have become an artacceptable means of creating murine models of HCV infection (Knetman &Mercer (2005) Hepatology 41:703-706; Kneteman & Toso (2009) Methods Mol.Biol. 510:383-399; Grompe, et al. (1999) Semin. Liv. Dis. 19:7-14). Thisapproach involves transplanting primary human hepatocytes intoimmunodeficient mice which have a lethal defect in their own endogenoushepatocytes. As the endogenous mouse hepatocytes die, transplantedprimary human hepatocytes can repopulate the mouse liver, resulting inmice with chimeric human livers permissive for HCV infection.

Therefore, to demonstrate in vivo activity of ezetimibe, experiments areperformed using mice triply mutant for Fah, Rag2 and the common γ-chainof the interleukin receptor (Fah^(−/−)/Rag2⁻⁻/Il2rg^(−/−) mice), whichhave been shown to be readily permissive for hepatic xenorepopulation(Azuma, et al. (2007) Nat. Biotechnol. 25:903-910; Schultz, et al.(2007) Nat. Rev. Immunol. 7:118-130). Alternatively, severe combinedimmunodeficient (SCID)/urokinase plasminogen activator (uPA) mice can beused. Mice are transplanted with primary human hepatocytes according toconventional methods (Azuma, et al. (2007) Nat. Biotechnol. 25:903-910;Bissig, et al. (2007) Proc. Natl. Acad. Sci. USA 104:20507-20511) andmonitored for two months post-transplantation to assess repopulationefficiency by measuring human albumin serum levels. Using these models,mice can be assessed for the prevention of HCV infection or theinhibition of an already established infection.

To demonstrate prevention, mice are treated with either 1) ezetimibe ata dose of 10 mg/kg/day via oral gavage or 2) a diluent control via oralgavage approximately 7 days prior to infection with HCV. Mice areinjected with HCV positive human serum (of any HCV genotype) and HCV RNAserum levels are quantified daily and/or weekly to assess the efficacywith which the treatments reduces the amplification of HCV RNA levels.Evidence that ezetimibe reduces the amplification HCV levels in vivo, ascompared to untreated animals, provides in vivo evidence that ezetimibeis an effective treatment for HCV in patients. In this respect, it isexpected that ezetimibe prevents the establishment of HCV infection.

To demonstrate treatment, mice are infected with HCV positive humanserum (of any HCV genotype). HCV RNA serum levels are assessed routinelyto confirm infection and monitor the establishment of a steady-statechronic infection in vivo. Groups of chronically HCV-infected chimericmice are then treated in parallel with either (1) a type I interferon(e.g., PEGASYS at 30 μg/kg twice weekly via subcutaneous injection), (2)ezetimibe alone at a dose of 10 mg/kg/day via oral gavage, or (3) acombination of both type I interferon (e.g., PEGASYS at 30 μg/kg twiceweekly) and ezetimibe (10 mg/kg/day). During the treatment period, HCVRNA serum levels are quantified weekly to assess the efficacy with whichthe treatments reduce steady-state HCV RNA levels. Evidence thatezetimibe alone or in combination with IFN reduces HCV levels in vivo,provides in vivo evidence that ezetimibe is an effective treatment forHCV in patients. In this respect, it is expected that ezetimibe incombination with a type I interferon will synergistically enhanceclearance of HCV infection when compared to type I interferon orezetimibe alone.

EXAMPLE 5 Clinical Analysis of Ezetimibe

Studies have indicated that interferon and ribavirin work by blockingHCV production and upregulating the host immune response to produceclearance of infected hepatocytes (Neumann, et al. (1998) Science282:103-107). Small molecules are in development, which target the HCVNS3 protease and the viral polymerase in an effort to further improvethe efficacy of antiviral therapy. However, to date, no FDA-approvedtreatment has been shown to block uptake of HCV into uninfected livercells. An agent that inhibits de novo infection of hepatocytes would notonly prevent initial spread of the virus in the liver, but also becomplementary to the available medications and could have a dramaticimpact on sustained Virologic Response (SVR) rates (i.e., viralclearance) and serve to prevent or slow infection of the new liverpost-transplantation.

To compare safety and efficacy of the combination ezetimibe, interferon,and ribavirin with the standard of care derived from interferon andribavirin for the treatment of hepatitis C infection in humans, clinicalstudies are carried out. Subjects selected for the trial are patientswho are due to receive routine treatment for HCV with interferon plusribavirin. Using a prospective randomized study design with two arms,patients are randomized 1:1 to treatment with ezetimibe vs. noezetimibe. All patients receive interferon plus ribavirin per a typicalprotocol. Group 1 receives ezetimibe, 10 mg per day, for 14 days priorto interferon and ribavirin to assess tolerability and to assess impactof ezetimibe monotherapy on HCV RNA level. Participants are then givenezetimibe, 10 mg per day, during the first three months of interferonand ribavirin therapy. Group 2 receives only the standard of care fortreatment of HCV, i.e., interferon and ribavirin.

All subjects have the normal routine laboratory monitoring performed forpatients receiving treatment for hepatitis C. Specifically, baseline HCVRNA level and genotype are assessed and HCV RNA levels are measured atweek 4, 12, 24, end of treatment, and 24 weeks post-treatment. Inaddition, for the purposes of the study, HCV RNA levels are assessed onday −14, 0, 2, 7, and 14. The primary study endpoint for assessment ofefficacy of drug treatment is the difference in serum HCV RNA decreaseas well as proportion of patients with undetectable HCV RNA at week 4 ofantiviral therapy. Specifically, the degree of serum HCV RNA decrease inGroup 1 is compared to Group 2 to assess whether the addition ofezetimibe to the standard of care enhances HCV inhibition and inparticular whether the addition of ezetimibe to the standard of careincreases the HCV clearance rate. The secondary endpoint is the slope ofHCV RNA decline over the first two weeks in Group 1 versus Group 2, asthis has proven to be an early predictor of SVR.

Similar clinical studies can be carried out to assess the efficacy andsafety of ezetimibe and interferon compared to interferon alone, as wellas assessing ezetimibe in preventing infection in liverpost-transplantation. It is expected that in each case, i.e.,ezetimibe/interferon/ribavirin and ezetimibe/interferon, ezetimibe willenhance the clearance of the HCV infection as compared tointerferon/ribavirin or interferon alone.

1. A method for reducing or preventing entry of a hepatic virus into acell comprising contacting a cell with an effective amount of aNiemann-Pick C1-like 1 protein antagonist so that entry of hepatic virusinto the cell is reduced or prevented.
 2. The method of claim 1, whereinthe Niemann-Pick C1-like 1 protein antagonist is an azetidinone-basedcholesterol absorption inhibitor.
 3. The method of claim 2, wherein theazetidinone-based cholesterol absorption inhibitor is ezetimibe, or aderivative thereof.
 4. A method for preventing a hepatic virus infectioncomprising administering to a subject in need of treatment an effectiveamount of a Niemann-Pick C1-like 1 protein antagonist so that entry ofhepatic virus into cells is reduced or prevented thereby preventing ahepatic virus infection.
 5. The method of claim 4, wherein the subjecthas had a liver transplant.
 6. The method of claim 4, wherein theNiemann-Pick C1-like 1 protein antagonist is an azetidinone-basedcholesterol absorption inhibitor.
 7. The method of claim 5, wherein theazetidinone-based cholesterol absorption inhibitor is ezetimibe, or aderivative thereof.
 8. A synergistic composition for treating a hepaticvirus infection comprising a Niemann-Pick C1-like 1 protein antagonistand at least one other agent that inhibits hepatic virus infection, withthe proviso that the other agent is not simvastatin.
 9. The synergisticcomposition of claim 8, wherein the Niemann-Pick C1-like 1 proteinantagonist is an azetidinone-based cholesterol absorption inhibitor. 10.The synergistic composition of claim 9, wherein the azetidinone-basedcholesterol absorption inhibitor is ezetimibe, or a derivative thereof.11. The synergistic composition of claim 8, wherein the at least oneother agent that inhibits hepatic virus infection is a type Iinterferon.
 12. The synergistic composition of claim 11, wherein thetype I interferon (IFN) is IFN-α or IFN-β.
 13. The synergisticcomposition of claim 8, further comprising a pharmaceutically acceptablecarrier.
 14. A method for effecting clearance of hepatic virus from acell comprising contacting a cell infected with a hepatic virus with aneffective amount of a synergistic composition including a Niemann-PickC1-like 1 protein antagonist and at least one other agent that inhibitshepatic virus infection so that the hepatic virus is cleared.
 15. Themethod of claim 14, wherein the Niemann-Pick C1-like 1 proteinantagonist is an azetidinone-based cholesterol absorption inhibitor. 16.The method of claim 15, wherein the azetidinone-based cholesterolabsorption inhibitor is ezetimibe, or a derivative thereof.
 17. Themethod of claim 14, wherein the at least one other agent that inhibitshepatic virus infection is a type I interferon.
 18. The method of claim17, wherein the type I interferon (IFN) is IFN-α or IFN-β.
 19. A methodfor treating a hepatic virus infection comprising administering to asubject with a hepatic virus infection an effective amount of asynergistic composition including a Niemann-Pick C1-like 1 proteinantagonist and at least one other agent that inhibits hepatic virusinfection so that the hepatic virus infection is treated.
 20. The methodof claim 19, wherein the Niemann-Pick C1-like 1 protein antagonist is anazetidinone-based cholesterol absorption inhibitor.
 21. The method ofclaim 20, wherein the azetidinone-based cholesterol absorption inhibitoris ezetimibe, or a derivative thereof.
 22. The method of claim 19,wherein the at least one other agent that inhibits hepatic virusinfection is a type I interferon.
 23. The method of claim 22, whereinthe type I interferon (IFN) is IFN-α or IFN-β.